The exocytotic release of catecholamines from chromaffin cells of the adrenal medulla has important cardiovascular and metabolic effects, and, in addition, serves as a model for secretion of other pre-packaged hormones and neurotransmitters. Using primary dissociated cultures of bovine adrenal medullary chromaffin cells, I will explore possible biochemical and physiological mechanisms which play a role in exocytotic release. I will examine 1) the relationship to secretion of intracellular Ca ion-dependent phosphorylation of specific protein and phospholipids, 2) the role in secretion of chemiosmotic and osmotic mechanisms involving chromaffin granules, and 3) the role in secretion of cell surface carbohydrates. Intracellular phosphorylation will be studied by incubating cells in (32P) phosphate to label intracellular high energy phosphate stores. In preliminary studies we found that carbachol stimulated Ca ion-dependent phosphorylation of 2 specific proteins. The phosphorylation of one of them was closely correlated with catecholamine secretion. It will be determined whether the primary stimulus for phosphorylation is cholinergic receptor occupancy, membrane depolarization, or Ca ion influx. The subcellular localization will be investigated and the biochemistry of the proteins in cell homogenates will be explored. Cholinergic agonists also stimulated phosphorylation of phosphatide acid and phosphatidylinositol. The relationship of lipid phosphorylation to secretion will also be investigated. ATP, Mg ions and Cl ion regulate chemiosmotic processes in purified chromaffin granules, the intracellular storage vesicles in chromaffin cells. The intracellular concentrations of these constituents will be measured, and the effects on exocytosis of chloride and other anions in the medium will be explored. The effects on secretion of manipulations designed to alter chemiosmotic processes of intracellular granules will be examined with careful attention given to possible changes in energy metabolism. The ability of various lectins to induce or alter secretion will be investigated. Lectins will also be used to determine if membrane glycoproteins from the inner surface of chromaffin granules appear on the cell surface during secretion.